In vivo fluorescence macro- and microscopic imaging is increasingly being used for clinical disease diagnosis and small animal research. In order to extend fluorescence imaging for a wide range of basic and clinical applications, it may be preferable to utilize flexible, miniaturized endoscopes. The performance of high quality fluorescent imaging procedures through a miniature flexible probe may be difficult due to the inability to incorporate a rapid beam scanning mechanism at the distal end of miniature probes and the limited number of optical fibers that can fit within the confines of small diameter fiber-optic imaging bundles.
Conventional procedures which apparently implemented fluorescence imaging through probes with a diameter of less than 2 mm have been performed using fiber optic bundles. For example, probes which vary in diameter from 600 μm to 1.8 mm have been used to obtain images of vessels in the mouse cremaster muscle, and which visualized labeled circulating cells. (See E. Laemmel et al., “Fibered confocal fluorescence microscopy (Cell-viZio™) facilitates extended imaging in the field of microcirculation—A comparison with intravital microscopy,” J. Vasc. Res., Vol. 41(5), 400 (2004)). As described in this publication, images of cells labeled with Fluorescein Isothiocyanate (“FITC”) (e.g., excitation with 488 nm) were obtained at 12 Hz with a maximal field of view of 400 μm×280 μm through probes with ˜10,000 optical fibers.
An 800 μm diameter endoscope with 10,000 optical fibers which can be used with Cy5.5 and Cy7, excited at 673 nm can also be utilized. (See M. A. Funovics et al., “Miniaturized multichannel near infrared endoscope for mouse imaging,” Molecular Imaging, Vol. 2(4), 350 (2003)). The imaging tip, which has a 56° field of view in water, can also facilitate white light reflectance imaging with a resolution of 7 line pairs per millimeter, as determined with an USAF 1951 resolution target. Exemplary images were presented from mouse vasculature and of protease activity in an ovarian tumor with rates ranging from 3 to 10 Hz. (See M. A. Funovics et al., “Catheter-based in vivo imaging of enzyme activity and gene expression: Feasibility study in mice,” Radiology, Vol. 231(3), 659 (2004)). According to this publication, tumors expressing green fluorescent protein were also observed.
Spectral encoding has been previously demonstrated for reflectance imaging. (See G. J. Tearney et al., “Spectrally encoded confocal microscopy,” Opt. Lett., Vol. 23(15), 1152 (1998); and G. J. Tearney et al., “Spectrally encoded miniature endoscopy,” Optics Letters, Vol. 27(6), 412 (2002)). In this exemplary technique, broadband light from an optical fiber may be dispersed by a grating, and focused onto a line on the sample. In this matter, the image does not have to be scanned in this dimension. A reflected light returns through the lens, grating, and optical fiber and the spectrally encoded image is then decoded via heterodyne Fourier transform spectroscopy (see G. J. Tearney et al., “Spectrally encoded confocal microscopy,” Opt. Lett., Vol. 23(15), 1152 (1998)) or with another grating in conjunction with a CCD detector (see G. J. Tearney et al., “Spectrally encoded miniature endoscopy,” Optics Letters, Vol. 27(6), 412 (2002)).
The transverse dimension can then be scanned by, for example, rotating the fiber and distal optics, which can be implemented in small diameter probes. (See G. J. Tearney et al., “Scanning single-mode fiber optic catheter-endoscope for optical coherence tomography,” Opt. Lett., Vol. 21(7), 543 (1996)). Using this conventional technique, the number of resolvable points (n) along one spectrally encoded line can be determined by the spectral bandwidth (Δλ), center wavelength (λ0), beam diameter (d), and grating:
                              n          ≅                                    Δλ              ⁢                                                          ⁢              dG                                                      λ                0                            ⁢                              cos                ⁡                                  (                                      θ                    i                                    )                                                                    ,                            (        1        )            where G and θi are the grating groove density and incidence angle, respectively. (See G. J. Tearney et al., “Spectrally encoded miniature endoscopy,” Optics Letters, Vol. 27(6), 412 (2002)).
The spectrally encoded photoluminescient techniques are generally based on a similar concept. In this exemplary embodiment, the fluorescence emission may be Stokes shifted, and the spatial locations are generally no longer uniquely related to the detected wavelengths. As a result, spectroscopic methods and arrangements implementing the same may not be effective for decoding the image. In order to recapture the spatial information, a spectral-and-frequency-encoded (“SFE”) imaging techniques can utilize a wavelength-dependent frequency modulation of the excitation light before it is dispersed onto the sample via the grating. The fluorescence emission at each location can therefore be modulated in concert with the frequency of the excitation light, thereby producing an additional level of encoding.
Accordingly, it may be beneficial to address and/or overcome at least some of the deficiencies described herein above. For example, the reference interferometer signal could be used for active feedback control to correct non-linear movement of the scanning mirrors, thereby eliminating the need for post-acquisition processing.